1. Field of the Invention
The present invention relates to animals harboring a non-native germ cell, to corresponding animal lines and germ cells, and to methods for obtaining the same.
2. Discussion of the Background
There have been many attempts to influence differentiation of developing cells by modifying the genotype of an embryo and then observing its effect on the phenotypic development pattern in the progeny. These techniques included transgenic methods (Brinster et al, Harvey Lectures, 80:1-38 (1986) nuclear transfer (McGrath & Solter, Science (1983) 220:1300-1302) and cell-egg fusions (Graham, "Heterospecific Genome Interaction," 1969 Wistar Institute Press, pp. 19-35, ed. Defendi). Of these, the latter two have had limited success.
Another approach might be to add a stem cell(s) to an early embryo and determine its effect on development. For example, one might imagine that a stem cell from bone marrow would contribute to the population of, and thus modify the differentiation of the evolving bone marrow cells in the host embryo. To pilot these experiments, older embryo cells were introduced into young embryos resulting in modest success with colonization (Moustafa & Brinster, J. Exp. Zool. (1972) 181:193).
These initial experiments were followed by studies using bone marrow stem cells and teratocarcinoma stem cells (also called embryonal carcinoma cells). There was evidence that both these cells colonized the developing embryo (Brinster, J. Exp. Med. (1974) 140:1049-1056). In the case of the embryonal carcinoma (EC) cell, the colonization was demonstrated dramatically by a change in the color of hairs in the coat of the mouse. This was an exciting result which stimulated a great deal of interest among scientists in the field, because it showed the possibility of colonizing an animal with non-embryo cells. This would provide a means to introduce new genetic information through the DNA of the colonizing cells.
The next year these results were confirmed and extended by two other laboratories (for work done in one of these laboratories, see Mintz & Illmensee, Proc. Nat. Acad. Sci. (USA) (1975) 72:3585-3589) and it was demonstrated that the introduced EC cells may colonize numerous tissues including germ cells (sperm and eggs). Thus, a gene that was mutated, modified, or added to the cell in vitro could eventually end up in sperm or eggs of an animal, creating a new genetic strain of mice.
Unfortunately EC cells colonized the germline poorly and a better cell line was sought. In 1981 two scientists, Gail Martin and Martin Evans, independently described a more efficient cell designated the embryonic stem (ES) cell (Martin, Proc. Nat. Acad. Sci. (USA) (1981) 78:7634; Evans & Kaufman, Nature (1981) 292:154). These cells colonize the germline better than EC cells. However, it seems likely that they arise from the same pool of primitive cells in the embryo and are quite similar in biological characteristics.
Embryonic stem cells can be modified in vitro (in culture flasks) by adding genes or changing endogenous genes and then the modified cells introduced into a blastocyst where they participate in development and can become sperm. This technique allows very specific modification of the mouse genome and perhaps other species.
Techniques for obtaining non-human transgenic animals through the injection of DNA into eggs are also known. See e.g., Gordon et al, Proc. Nat. Acad. Sci. (USA), (1980) 77:7380-7384. These techniques however, as well as the use of ES cells noted above, are very labor intensive.
Gene therapy is by definition the insertion of genes for the purpose of medicinal therapy. The principle underlying gene therapy is to, rather than deliver doses of one or more pharmacologic molecule, deliver a functional gene whose RNA or protein product will produce the desired biochemical effect in the target cell or tissue. The genes may be delivered into endogenous cells or within new cells delivered to the animal. There are several potential advantages of gene therapy over classical biochemical pharmacology, including the fact that inserted genes can produce extremely complex molecules, including RNA and proteins, which can be extraordinarily difficult or impossible to administer and deliver themselves.
The many applications of gene therapy, particularly via stem cell genetic insertion, have been extensively reviewed (see, Boggs et al., Int. J. Cell Cloning (1990) 8, 80; Kohn et al., Cancer Invest. (1989) 7, 179; Lehn, Bone Marrow Transpl. (1990) 5. 287; and Verma et al., Scientific Amer. (November 1990) 68). Genetically transformed human stem cells have wide potential application in clinical medicine, as agents of gene therapy.
Methods and compositions are known for the ex vivo replication and stable genetic transformation of animal, including human, stem cells and for the optimization of such stem cell cultures. The applications of gene therapy could be advantageously expanded if techniques were available for the application of gene therapy to animal primitive germ cells.